In article <slrn453m6g8.atl.dek at socrates.ucsf.edu>, dek at socrates.ucsf.edu
says...
>Hi, folks. I've been working on doing a miniprep of overnight
>cultures for a few weeks and have been having some technical difficulties.
>Nobody in the lab is able to figure out why this is happening.
>>I've created a plasmid vector using ligation which I transform into
>DH5-alpha cells.
[snip - 12 kb DNA present in non-transformed bacteria]
Are your "DH5-alpha" actually DH5-alpha-F'? There was a series of
posts recently that suggested the F factor may carry through with some
methods of plasmid purification so this may be the source of the mystery
band.
>As far as I can tell I am following the miniprep procedure (cell pellet
>into 100ul GTE, resuspend, add 200ul alkali lysis solution, on ice 5 min,
>add 150ul 3M NaOAc, on ice 5min, spin down, keep supernatant, do
>ethanol precip and raiise up DNA pellet in TE.)
This is a fairly crude method (one I use all the time for check digests)
and I also see high MW material stained and I ignore it. I would only
worry if I saw this after eg. CsCl purification. One extra step that can
clear up the pattern is a quick digestion with RNase (or just add a
little to your restriction digests).
If you want to make sure that it is the F factor then try your
miniprep procedure on some bona fide DH5-alpha-F-minus bacteria.
Otherwise just concentrate on the good clones and have a closer look
after you have done a clean maxiprep.
Bernard
Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)
Postdoc for sale: Will create transgenic mice in return for food.